Perfusion airlift bioreactor

ABSTRACT

A perfusion airlift bioreactor system that consists of the bioreactor, a media reservoir and a molecular weight cutoff (MWCO) filter positioned between the media reservoir and the airlift bioreactor.

BACKGROUND OF THE INVENTION

The present invention relates to an improved bioreactor apparatus usefulfor eukaryotic cell culture, where cell density and bioproduct areincreased by, using a molecular weight cutoff filter. The apparatus ofthe present invention utilizes design features which afford optimumagitation of the cells with minimum mechanical shear force. This isaccomplished by utilizing a gas feed to provide gentle aeration andmixing. The novel feature of the invention is the use of an externalmolecular weight cutoff (MWCO) filter to perfuse nutrients in and wasteproducts out of the bioreactor while maintaining cells and theirproducts within the bioreactor.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide anairlift bioreactor capable of increasing both cell density andbioproduct/s concentration in the airlift bioreactor.

In keeping with this object and the others which will become apparenthereinafter one feature of the airlift bioreactor system is the use ofan external molecular weight cutoff filter to perfuse nutrients in andwaste products out of the bioreactor while maintaining cells and cellproducts within the bioreactor. This external cross flow filter isattached to both the bioreactor (the lumen side of the filter) and themedia reservoir (shell space of the filter). When culture supernatantand reservoir media are both pumped through the filter, waste productsdiffuse from the culture and nutrients diffuse into the culture. Thisallows the cells to achieve a significantly higher density and attendantaccumulation of products in this culture supernatant. The increases incell density and bioproduct/s productivity are proportional to the ratioof the volume of the medium in the reservoir to that of the bioreactor.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view showing a vertical cross section of theapparatus.

FIG. 2 is a graph showing improved cell density when compared to a T-75flask.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

An arrangement for growing micro-organisms is shown in FIG. 1 with theairlift bioreactor (10) of the molecular weight cutoff filter (11) amedia reservoir (12) and pumps (13) and (14) which perfuse the mediathrought the molecular weight cutoff filter. The airlift bioreactor is avessel having a volume of about 2 liters that includes a cylindricaldraft tube (15). An inlet tube (17) is connected to a suitable pump andfilter (not shown) for supplying gases (16) for stirring and oxygenationto the medium contained therein. The pump (13) circulates the medium inthe bioreactor through the tube (20) and into and out of the bioreactorthrough apertures (18) and (19). The pump (14) circulates the mediumfrom the storage vessel (12) through the tubes (21) and into and out ofthe vessel through the apertures (22) and (23).

The invention is illustrated by the following example which illustratesthe advantages of the present invention.

Hybridomas were grown in a series 500 2 liter airlift bioreactorfurnished by LH Fermentation (a division of Porton International, Inc.)of Hayward, Calif. The operational volume of airlift bioreactor wasabout 2.3 liters. For comparison, conventional tissue culture wasperformed in a T-75 flask in a humidified carbon dioxide incubator.

The bioreactor was sparged with a mixture of carbon dioxide, oxygen andair at a rate of about 20 ml per minute. The pH was maintained at7.0±0.1, the temperature at 37±1° C. and oxygen was maintained at airsaturation. Hybridoma and medium from the airlift bioreactor and freshmedium from the 50 liter reservoir were perfused through a 19 squarefoot 30,000 molecular weight cutoff (MWCO) hollow fiber bundle. Thecells were grown in a growth medium RPMI-1640 supplemented with 5% fetalbovine serum furnished by J.R. Scientific (a division of PortonInternational, Inc.) of Woodland, Calif.

The perfusion system incorporating a 2 liter airlift bioreactor and a 50liter reservoir shown in FIG. 1 resulted in a 15 fold increase in celldensity with excellent viability and a 21 fold increase in monoclonalantibody (MAb) concentration in the airlift bioreactor when comparedwith conventional batch culture using a T-75 flask. The data collectedis set out in Table 1 below:

                  TABLE 1                                                         ______________________________________                                                                  MONOCLONAL                                                     MAXIMUM        ANTIBODY                                                       CELL DENSITY   CONCENTRATION                                       BIOREACTOR (× 10.sup.6 live cells/ml)                                                             (micrograms/ml)                                     ______________________________________                                        T-75 flask 0.55           5.6                                                 Perfusion airlift                                                                        8.10           115.0                                               bioreactor                                                                    ______________________________________                                    

The data is shown graphically in FIG. 2. The lines with the points drawnas triangles shows hybridoma density when the 2 liter airlift and 50liter reservoir perfusion system was used with a 19 square foot 30,000MWCO hollow fiber bundle. The perfusion took place at an average of 40ml per minute of bioreactor and reservoir medium through the filter. TheMAb concentration in micrograms per ml was 115 as set out in the Tableabove.

The line with the points drawn as circles shows conventional batchgrowth curve in a T-75 flask with MAb productivity of only 5.6micrograms per ml.

It is apparent from the data that there is a 21 fold increase in MAbconcentration in the airlift bioreactor when compared to a conventionalT-75 flask batch culture.

The increased concentration of MAb in a bioreactor renders purificationsimpler. The ratio of MAB to non-MAb proteins is increased from 0.28 to6% in the perfusion airlift bioreactor for this low MAb producinghybridoma. The total antibody productivity for a 2 liter airliftperfusion culture is equal to that of a 50 liter airlift bioreactoroperated in a batch mode.

Without further analysis the foregoing will fully reveal the gist of thepresent invention so that others can be applying current knowledge canreadily adapt it for various applications without omitting features thatfrom the standpoint of prior art clearly constitute essentialcharacteristics of the generic or specific aspects of this invention.

What is claimed is:
 1. An apparatus for growing eukaryotic cells consisting essentially of:(a) a two liter airlift bioreactor accomaditing eukaryotic cells in suspension, encapsulated or on microcarriers in a medium supporting cell growth, (b) a 19 square foot 30,000 molecular weight cutoff hollow fiber bundle filter, positioned between said bioreactor and said media reservoir, (c) pump means operably connected to said bioreactor and said filter for circulating said medium into the filter and returning said medium to the bioreactor, (d) pump means operably connected to said filter and a 50 liter medium reservoir for circulating said medium from said reservoir to said filter and returning said medium to said medium reservoir, (e) pump means operably connected to said bioreactor for admitting air into said airlift bioreactor for stirring the suspension of cells and medium whereby, (f) the spent bioreactor medium containing product/s is dialyzed aganst a fresh reservoir medium through said hollow fiber filter. 